The present invention relates to a novel linker based solid support for use in peptide synthesis and combinatorial library synthesis, methods for generating the linker moiety on the solid support, and methods of using the supports to synthesize peptides and small molecule combinatorial libraries. The new solid support of this invention also comprises a functionalized linker covalently attached to the solid supports such as polystyrene.
Rapid development of solid phase synthesis calls for the development of new and versatile solid support to meet the newer synthetic challenges. The solid supports for the synthesis of peptides have been very well established and even synthesis of cyclic peptides, peptide amides, peptide aldehydes etc are now possible directly on the support [(a) Barany, G. et al., Int. J. Peptide protein Res. 30, 705-739, 1987.; (b) Fields, G. B. et al., Int. J. Peptide protein Res. 35, 161-214, 1990. (c) Lloyd-Williams, P. et al., Tetrahedron, 49, 11065-11133, 1993 (d) Wang, S. S., J. Amer. Chem. Soc. 95, 1328, 1973 (e) Barlos, K., et al., Tetrahedron Letters, 30, 3947, 1989 (f) Beebe, X., et al., J. Org. Chem., 60, 4204, 1995 (g) Rink, H., Tetrahedron Letters, 28, 3787, 1987 (h) Rapp, W., et al., in xe2x80x9cPeptides 1988xe2x80x9d, Proc. 20th European Peptide Symposium, Jung G. and Boyer E. (Eds.), Walker de Gruyter, Berlin, pp 199 1989]. Further, synthesis of small organic molecules require more versatile and broad range solid supports. A number of supports are reported in the literature with a variety of functionalities to attach the synthons as well as their stability and susceptibility towards the synthetic reagents and experimental conditions used [Oesapay, G., et al, in xe2x80x9cPeptides Chemistry, Structure and Biology, Proc. 13th American Peptide Symposiumxe2x80x9d, Hodges, R. S. and Smith J. A. (Eds.), ESCOM, Leiden, pp 435, 1994, (b) Hermkens, P. H. H. et al, Tetrahedron, 52, 4527, 1996 (c) Hermkens, P. H. H. et al, Tetrahedron, 53, 5643, 1997 (d) Brown, R. C., J. Chem. Soc. Perkin-I, 3293, 1998]. Mostly, the available supports require drastic conditions for the cleavage of molecules from the solid support involving strong acids, which are harmful to the molecules. Further, aminolysis and reductive cleavage methods result in side reaction and racemization, particularly in the case of peptides. On the other hand, supports, which require relatively milder conditions, do not withstand various reagents and experimental conditions used in organic synthesis, particularly in the case of small organic molecules. Therefore, development of new and versatile supports, which are orthogonal compatible, will be highly beneficial.
Development of various supports essentially based on the strategy to incorporate a bi-functional linker between the solid matrix and growing molecule are in vogue. The first step involves attachment of linker molecule on the solid matrix and subsequently the desired synthetic scaffold is constructed on the linker bearing solid support. This allows utilization of the desired functionalities for the attachment of synthetic scaffold and to achieve desired chemical properties on the solid support. Based on this premise a number of linker based solid supports have been developed and are in commercial use [Oesapay, G., et al, in xe2x80x9cPeptides Chemistry, Structure and Biology, Proc. 13th American Peptide Symposiumxe2x80x9d, Hodges, R. S. and Smith J. A. (Eds.), ESCOM, Leiden, pp 435, 1994, (b) Hermkens, P. H. H. et al, Tetrahedron, 52, 4527, 1996 (c) Hermkens, P. H. H. et al, Tetrahedron, 53, 5643, 1997 (d) Brown, R. C., J. Chem. Soc. Perkin-I, 3293, 1998. And a) Blackburn, C. Biopolymers, peptide sciences, 47, 311, 1998 (b) Patek, M. and Lebl, M. ibid. 353 (c) Barany, G., et al, U.S. Pat. No.: 5,235,028 1993.(d) Barany, G., U.S. Pat. No.: 5,306,562, 1994 (e) Jensen, K. J., et al., U.S. Pat. No. 5,917,015, 1999].
Working on similar lines, the applicants also developed earlier a linker for solid phase synthesis of peptides [Katti, S. B. et al, J. Chem. Soc. Chem. Commun., 843-844, 1992]. The base labile linker as shown in FIG. II, formula VII was prepared using readily available raw materials and synthesized for each amino acid separately to be used at C-terminal. It was prepared according to the method used for the synthesis of PAM resin [Mitchell, A. R. et al., J. Org. Chem. 43, 2845, 1978]. This provides pre-loaded amino acid with linker on solid support for further synthesis of peptides. The synthesis started with the treatment of chloroacetic acid (I) with phenacyl bromide (II) to obtain corresponding ester (III). The ester III was treated with mercaptoethanol (IV) to obtain protected linker (V). The hydroxy group of the linker was acylated with suitably protected amino acid (Boc or Fmoc) with DCC and DMAP followed by the deblocking of phenacyl ester by Zn/AcOH to obtain free acid (VI). The sulfide group of the free acid VI was then oxidized to obtain corresponding sulphone (VII) by the treatment with oxone. The linker acid carrying the suitably protected amino acid was loaded on the amino methyl resin by DIC/HOBT procedure and quantitative loading was obtained. Having loaded the linker bearing first amino acid on solid support, the utility of linker based solid support was demonstrated by synthesis of peptides using Boc and Fmoc chemistry employing the known procedures. The cleavage of the peptide from the solid support was carried out under extremely mild conditions by treatment with dioxane:methanol:4N NaOH (30:9:1) for 30 min. This provides an opportunity to obtain protected peptide fragments, which are quite useful intermediates for the synthesis of large peptides and proteins [Kaiser, E. T., Acc. Chem. Res. 22, 47, 1989; Williams, P. et al. Tetrahedron, 49, 11065, 1993]. Thus, it was demonstrated that the linker was compatible with both, Boc and Fmoc chemistry protocols and can be synthesized easily in laboratory. Using this linker several peptides and protected peptide fragments have been synthesized.
In spite of the various advantages, there are certain limitations with the linker based solid support mentioned above. For the synthesis of peptides the linker with the amino acid at the C-terminus is to be synthesized separately for each synthesis [FIG. II shown in the accompanying drawings]. Therefore, the synthesis for each sequence requires additional steps and longer time. Further, the linker having protected amino acid is oxidized using oxone, a powerful oxidizing agent, before loading it on the solid support, therefore, amino acids, prone to oxidation can not be used as the first amino acid. Thus peptide sequences having cystein, methionine, tryptophan and tyrosine at the C-terminus pose problems as these amino acids are oxidized during the oxone oxidation. Secondly, it has been observed that the linker of formula VII, as shown in FIG. II of the accompanying drawings is not completely stable under the cleavage conditions used for the removal of Fmoc group, particularly for the synthesis of longer peptides. It was observed that prolonged exposure with 20% piperidine solution resulted in the cleavage of growing chain from the solid support. Since the cleavage of products from the solid support occurs via xcex2-elimination, the basicity of the 20% piperidine solution is enough to cause the xcex2-elimination of the linker and resulted in the partial cleavage of the peptide at each deblocking step.
The main objective of the present invention is to provide an orthogonally compatible linker based solid support for organic synthesis.
Another objective of the invention is to provide a new linker based solid support for peptide synthesis employing Boc and Fmoc chemistry protocols separately or in combination.
One more objective of the invention is to provide a new solid support comprising a functionalized linker covalently attached to the solid supports such as polystyrene.
Another objective of the present invention is to provide a method of synthesis of hydroxyethyl-sulphenyl-acetamido resin.
Still another objective of the invention is to provide a new solid support for the synthesis of protected peptide fragments for use in solid phase convergent synthesis of longer peptides.
Yet, another objective of the present invention is to enhance the scope of the new linker based solid support for the synthesis of small organic molecules to generate combinatorial libraries